After its release, the concentration of dopamine in and around the synapse is rapidly reduced by reuptake mechanisms. The dopamine transporter (DAT), the protein which carries out this reuptake process at the plasma membrane, is the major molecular target responsible for the rewarding properties and abuse potential of cocaine and related psychostimulants. DT requires extracellular Na+ and Cl- and couples the translocation of dopamine to the movement of these ions. Despite the recent cloning of DAT and related neurotransmitter transporters, the molecular structure and mechanism of transport by these transporters are poorly understood. The long-term goals of this project are to determine the structural bases of substrate translocation by DAT and of its inhibition by drugs such as cocaine. Our recent work indicates that extracellular and intracellular loop residues play a critical role in the structure and function of DAT. We have identified four endogenous cysteines in DAT, the reaction of which with polar sulfhydryl regents affects the binding of cocaine analogues. Two of these residues are in putative cytoplasmic loops, suggesting that these loops play a critical role in determining the structure of the binding site. Remarkably, the presence of cocaine or dopamine dramatically retards the reaction of polar sulfhydryl reagents with these cysteines, even in a membrane preparation in which the regents have direct access to intracellular residues. Therefore, binding of substrate or inhibitor alters the conformation and hence the accessibility of the cytoplasmic loops. In contrast, Cys90, the endogenous cysteine at the putative extracellular end of the first membrane-spanning segment becomes more accessible in the presence of cocaine, suggesting that a conformational change occurs in this region as well. Thus, we propose the following specific aims: 1) To determine the topology of DAT by ascertaining whether each putative loop is extracellular or intracellular. 2) To determine the functional role of the loop residues for cocaine and dopamine binding and for dopamine transport. 3) To determine approximately proximity between extracellular loops. These studies will allow us to infer critical information about the packaging of the membrane spanning segments, the arrangement of the loops and conformation changes in these loops which accompany binding and transport. This information will complement our ongoing studies, as well as the other components of the Program Project Grant, by providing a structural framework within which to organize the experimental results. It will also provide additional experimental data with which to test and refine molecular models of DAT and related transporters such as the serotonin transporter and norepinephrine transporters, which are targets for various antidepressant drugs.